Interpreting Prevotella and Bacteroides as biomarkers of diet and lifestyle.

BACKGROUND: In a series of studies of the gut microbiome, "enterotypes" have been used to classify gut microbiome samples that cluster together in ordination analyses. Initially, three distinct enterotypes were described, although later studies reduced this to two clusters, one dominated by Bacteroides or Clostridiales species found more commonly in Western (American and Western European) subjects and the other dominated by Prevotella more often associated with non-Western subjects. The two taxa, Bacteroides and Prevotella, have been presumed to represent consistent underlying microbial communities, but no one has demonstrated the presence of additional microbial taxa across studies that can define these communities. RESULTS: We analyzed the combined microbiome data from five previous studies with samples across five continents. We clearly demonstrate that there are no consistent bacterial taxa associated with either Bacteroides- or Prevotella-dominated communities across the studies. By increasing the number and diversity of samples, we found gradients of both Bacteroides and Prevotella and a lack of the distinct clusters in the principal coordinate plots originally proposed in the "enterotypes" hypothesis. The apparent segregation of the samples seen in many ordination plots is due to the differences in the samples' Prevotella and Bacteroides abundances and does not represent consistent microbial communities within the "enterotypes" and is not associated with other taxa across studies. The projections we see are consistent with a continuum of values created from a simple mixture of Bacteroides and Prevotella; these two biomarkers are significantly correlated to the projection axes. We suggest that previous findings citing Bacteroides- and Prevotella-dominated clusters are the result of an artifact caused by the greater relative abundance of these two taxa over other taxa in the human gut and the sparsity of Prevotella abundant samples. CONCLUSIONS: We believe that the term "enterotypes" is misleading because it implies both an underlying consistency of community taxa and a clear separation of sets of human gut samples, neither of which is supported by the broader data. We propose the use of "biomarker" as a more accurate description of these and other taxa that correlate with diet, lifestyle, and disease state.

Morphologic effects of estrogen stimulation on 3D MCF-7 microtissues.

In the development of human cell-based assays, 3-dimensional (3D) cell culture models are intriguing as they are able to bridge the gap between animal models and traditional two-dimensional (2D) cell culture. Previous work has demonstrated that MCF-7 human breast carcinoma cells cultured in a 3D scaffold-free culture system self-assemble and develop into differentiated microtissues that possess a luminal space. Exposure to estradiol for 7 days decreased lumen formation in MCF-7 microtissues, altered microtissue morphology and altered expression of genes involved in estrogen signaling, cell adhesion and cell cycle regulation. Exposure to receptor-specific agonists for estrogen receptor alpha, estrogen receptor beta and g-protein coupled estrogen receptor resulted in unique, receptor-specific phenotypes and gene expression signatures. The use of a differentiated scaffold-free 3D culture system offers a unique opportunity to study the phenotypic and molecular changes associated with exposure to estrogenic compounds.

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels.

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17ß-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.

Developmental exposure to estrogen alters differentiation and epigenetic programming in a human fetal prostate xenograft model.

Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure.

The stage-specific testicular germ cell apoptotic response to low-dose X-irradiation and 2,5-hexanedione combined exposure. I: Validation of the laser capture microdissection method for qRT-PCR array application.

Over the past decade, laser capture microdissection (LCM) has grown as a tool for gene expression profiling of small numbers of cells from tumor samples and of specific cell populations in complex tissues. LCM can be used to study toxicant effects on selected cell populations within the testis at different stages of spermatogenesis. There are several LCM-related hurdles to overcome, including issues inherent to the method itself, as well as biases that result from amplifying the LCM-isolated RNA. Many technical issues associated with the LCM method are addressed here, including increasing RNA yield and obtaining more accurate quantification of RNA yields. We optimized the LCM method optimized to generate RNA quantities sufficient for quantitative reverse transcription polymerase chain reaction (qRT-PCR) array analysis without amplification and were able to validate the method through direct comparison of results from unamplified and amplified RNA from individual samples. The addition of an amplification step for gene expression studies using LCM RNA resulted in a bias, especially for low abundance transcripts. Although the amplification bias was consistent across samples, researchers should use caution when comparing results generated from amplified and unamplified LCM RNA. Here, we have validated the use of LCM-derived RNA with the qRT-PCR array, improving our ability to investigate cell-type and stage-specific responses to toxicant exposures.

The stage-specific testicular germ cell apoptotic response to low-dose radiation and 2,5-hexanedione combined exposure. II: qRT-PCR array analysis reveals dose dependent adaptive alterations in the apoptotic pathway.

Testicular effects of chemical mixtures may differ from those of the individual chemical constituents. This study assessed the co-exposure effects of the model germ cell- and Sertoli cell-specific toxicants, X-irradiation (x-ray), and 2,5-hexanedione (HD), respectively. In high-dose studies, HD has been shown to attenuate x-ray-induced germ cell apoptosis. Adult rats were exposed to different levels of x-ray (0.5 Gy, 1 Gy, and 2 Gy) or HD (0.33%), either alone or in combination. To assess cell type-specific attenuation of x-ray effects with HD co-exposure, we used laser capture microdissection (LCM) to enrich the targeted cell population and examine a panel of apoptosis-related transcripts using PCR arrays. The apoptosis PCR arrays identified significant dose-dependent treatment effects on several genes, with downregulation of death receptor 5 (DR5), Naip2, Sphk2, Casp7, Aven, Birc3, and upregulation of Fas. The greatest difference in transcript response to exposure was seen with 0.5 Gy x-ray exposure, and the attenuation effect seen with the combined high-dose x-ray and HD did not persist into the low-dose range. Examination of protein levels in staged tubules revealed a significant upregulation in DR5, following high-dose co-exposure. These results provide insight into the testis cell-specific apoptotic response to low-dose co-exposures of model testicular toxicants.

Comparison of brush and biopsy sampling methods of the ileal pouch for assessment of mucosa-associated microbiota of human subjects.

BACKGROUND: Mucosal biopsy is the most common sampling technique used to assess microbial communities associated with the intestinal mucosa. Biopsies disrupt the epithelium and can be associated with complications such as bleeding. Biopsies sample a limited area of the mucosa, which can lead to potential sampling bias. In contrast to the mucosal biopsy, the mucosal brush technique is less invasive and provides greater mucosal coverage, and if it can provide equivalent microbial community data, it would be preferable to mucosal biopsies. RESULTS: We compared microbial samples collected from the intestinal mucosa using either a cytology brush or mucosal biopsy forceps. We collected paired samples from patients with ulcerative colitis (UC) who had previously undergone colectomy and ileal pouch anal anastomosis (IPAA), and profiled the microbial communities of the samples by sequencing V4-V6 or V4-V5 16S rRNA-encoding gene amplicons. Comparisons of 177 taxa in 16 brush-biopsy sample pairs had a mean R2 of 0.94. We found no taxa that varied significantly between the brush and biopsy samples after adjusting for multiple comparisons (false discovery rate ≤0.05). We also tested the reproducibility of DNA amplification and sequencing in 25 replicate pairs and found negligible variation (mean R2 = 0.99). A qPCR analysis of the two methods showed that the relative yields of bacterial DNA to human DNA were several-fold higher in the brush samples than in the biopsies. CONCLUSIONS: Mucosal brushing is preferred to mucosal biopsy for sampling the epithelial-associated microbiota. Although both techniques provide similar assessments of the microbial community composition, the brush sampling method has relatively more bacterial to host DNA, covers a larger surface area, and is less traumatic to the epithelium than the mucosal biopsy.

Differential response to abiraterone acetate and di-n-butyl phthalate in an androgen-sensitive human fetal testis xenograft bioassay.

In utero exposure to antiandrogenic xenobiotics such as di-n-butyl phthalate (DBP) has been linked to congenital defects of the male reproductive tract, including cryptorchidism and hypospadias, as well as later life effects such as testicular cancer and decreased sperm counts. Experimental evidence indicates that DBP has in utero antiandrogenic effects in the rat. However, it is unclear whether DBP has similar effects on androgen biosynthesis in human fetal testis. To address this issue, we developed a xenograft bioassay with multiple androgen-sensitive physiological endpoints, similar to the rodent Hershberger assay. Adult male athymic nude mice were castrated, and human fetal testis was xenografted into the renal subcapsular space. Hosts were treated with human chorionic gonadotropin for 4 weeks to stimulate testosterone production. During weeks 3 and 4, hosts were exposed to DBP or abiraterone acetate, a CYP17A1 inhibitor. Although abiraterone acetate (14 d, 75 mg/kg/d po) dramatically reduced testosterone and the weights of androgen-sensitive host organs, DBP (14 d, 500 mg/kg/d po) had no effect on androgenic endpoints. DBP did produce a near-significant trend toward increased multinucleated germ cells in the xenografts. Gene expression analysis showed that abiraterone decreased expression of genes related to transcription and cell differentiation while increasing expression of genes involved in epigenetic control of gene expression. DBP induced expression of oxidative stress response genes and altered expression of actin cytoskeleton genes.

Maturation of the developing human fetal prostate in a rodent xenograft model.

BACKGROUND: Prostate cancer is the most commonly diagnosed nonskin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. METHODS: We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate-specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. RESULTS: Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture microdissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30- and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. CONCLUSION: This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease.

Genomic minimalism in the early diverging intestinal parasite Giardia lamblia.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.